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WO2004004630A2 - Biomateriaux osteo-inducteurs - Google Patents

Biomateriaux osteo-inducteurs Download PDF

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Publication number
WO2004004630A2
WO2004004630A2 PCT/IB2003/002354 IB0302354W WO2004004630A2 WO 2004004630 A2 WO2004004630 A2 WO 2004004630A2 IB 0302354 W IB0302354 W IB 0302354W WO 2004004630 A2 WO2004004630 A2 WO 2004004630A2
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bone
osteogenic protein
molecular weight
composition
osteogenic
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WO2004004630A3 (fr
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Nicolaas Duneas
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Priority to AU2003240196A priority Critical patent/AU2003240196A1/en
Priority to JP2004519039A priority patent/JP2005536489A/ja
Priority to EP03732811A priority patent/EP1539812B1/fr
Priority to AT03732811T priority patent/ATE469916T1/de
Priority to CA002527619A priority patent/CA2527619A1/fr
Priority to DE60332848T priority patent/DE60332848D1/de
Priority to US10/518,723 priority patent/US7728116B2/en
Publication of WO2004004630A2 publication Critical patent/WO2004004630A2/fr
Publication of WO2004004630A3 publication Critical patent/WO2004004630A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/043—Mixtures of macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • A61L24/104—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
    • A61L24/04—Surgical adhesives or cements; Adhesives for colostomy devices containing macromolecular materials
    • A61L24/10—Polypeptides; Proteins
    • A61L24/108—Specific proteins or polypeptides not covered by groups A61L24/102 - A61L24/106
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/222—Gelatin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/26—Mixtures of macromolecular compounds

Definitions

  • THIS INVENTION relates to osteoinductive biomaterials.
  • the invention relates to an osteogenic composition and to the use of an osteogenic composition in therapy.
  • the osteogenic composition of the invention is particularly intended for human or mammalian tissue regeneration and for promoting or inducing bone growth.
  • osteoogenic protein refers to the material which is obtained by fractionation of total mammalian bone protein and which is capable of inducing bone formation.
  • the terms "osteogenic” and “osteoinductive” are considered to be synonymous.
  • Osteogenesis is the term used to describe the de novo formation of bone in adult mammals and is evidenced in adults during regeneration of bone fractures. It proceeds via a process which closely resembles embryonic osteogenesis.
  • Osteogenic protein contains, amongst other unidentified proteins, Bone Morphogenetic Proteins (BMPs). This is a family of characterized proteins which have been classified as part of the larger transforming growth factor- beta superfamily of morphogenic proteins. The BMP family comprises more than a dozen individual members which are known to be capable of inducing bone formation in mammals.
  • BMPs Bone Morphogenetic Proteins
  • a method of isolating osteogenic protein from bone in which an osteogenic protein containing fraction is extracted from bone and enriched by a sequence of enrichment steps selected from ultrafiltration and chromatography, there is provided the improvement of removing higher molecular weight components from the osteogenic protein containing fraction prior to the enrichment steps.
  • the higher molecular weight components may have a molecular weight of about 100 - 300 kDa.
  • the higher molecular weight components will typically include collagen, collagen fragments, collagen aggregates and mixtures thereof.
  • the method may include removing the higher molecular weight components by ultrafiltration.
  • the components may be removed by ultrafiltration through a 100 - 300 kDa nominal molecular weight membrane such as a 100 - 300 kDa nominal molecular weight polysulphone membrane.
  • the osteogenic protein containing fraction may be extracted from the bone using a chaotropic solution.
  • the chaotropic solution may contain urea, guanidinium chloride or combinations thereof.
  • the enrichment steps may include successive ultra-filtration of the osteogenic protein containing fraction through progressively smaller nominal molecular weight membranes followed by, or interspersed with, chromatographic enrichment steps.
  • the enrichment steps may thus include one or more chromatographic enrichment steps.
  • the osteogenic protein containing fraction may be concentrated and desalted through successive ultra-filtration steps.
  • the fraction may be concentrated and desalted through 10 kDA and 5 kDA ultra-filtration steps.
  • the chromatographic enrichment steps may be selected from one or more of heparin-sepharose chromatography, hydroxyapatite chromatography, reverse- phase silica chromatography and combinations of any two or more thereof.
  • a bone growth inducing composition which includes osteogenic protein, insoluble bone matrix (ICBM) and gelatin.
  • the composition may be in the form of a hydratable powder.
  • the insoluble bone matrix may be prepared by demineralizing whole bone powder with acid to produce an acid demineralised whole bone powder or matrix, extracting soluble components from the demineralised bone powder or matrix with a chaotropic agent such as aqueous urea or a guanidinium solution, water-washing the residue and then drying the residue to produce insoluble bone matrix.
  • a chaotropic agent such as aqueous urea or a guanidinium solution
  • the gelatin may be obtained by extracting insoluble bone matrix to produce a fraction rich in soluble collagen type I, precipitating the soluble collagen type I and drying the precipitate in vacuo.
  • the extraction may be with purified boiling water and the precipitation may be with ethanol.
  • the insoluble collagenous bone matrix may be mammalian. It may be non- human or human insoluble collagenous bone matrix. It is preferably human insoluble collagenous bone matrix or hlCBM.
  • the gelatin may accordingly be human gelatin.
  • the osteogenic protein may be prepared by a method as hereinbefore described.
  • the mass ratio between the osteogenic protein, the hlCBM and the human gelatin may be about 0,4 - 0,6 : 800 - 1200 : 100 - 1000.
  • the bone growth inducing composition may include the osteogenic protein in an amount of about 400 - 600 ⁇ g, the hlCBM in an amount of about 800 - 1200mg and the human gelatin in an amount of about 100 - 1000mg.
  • the bone growth inducing composition includes the osteogenic protein in an amount of about 500 ⁇ g, the hlCBM in an amount of about 1000mg and the human gelatin in an amount of about 200mg.
  • the invention extends to a bone growth inducing composition as described above in which the osteogenic protein is prepared by an improved method as described above.
  • a method of preparing a bone growth inducing composition including the steps of combining osteogenic protein, insoluble bone matrix and gelatin.
  • the insoluble collagenous bone matrix may be mammalian and may be selected from non-human and human insoluble collagenous bone matrix. It is preferably human insoluble collagenous bone matrix or hlCBM.
  • the gelatin may be human gelatin.
  • the osteogenic protein may be prepared by a method as hereinbefore described.
  • the osteogenic protein, the hlCBM and the human gelatin may be combined in a mass ratio of 0,4 - 0,6 : 800 :1200 : 100 - 1000.
  • the method may thus include combining the osteogenic protein in an amount of about 400 - 600 ⁇ g, the hlCBM in an amount of about 800 - 1200mg and the human gelatin in an amount of about 100 - 1000mg.
  • the method may include combining the osteogenic protein in an amount of about 500 ⁇ g, the hlCBM in an amount of about 1000mg and the human gelatin in an amount of about 200mg.
  • the method may include combining the osteogenic protein with the hlCBM in a dilute aqueous acidic solution, lyophilising the resulting mixture to produce a dry powder and mixing the powder with the human gelatin to produce a hydratable material.
  • a device for inducing bone growth in a mammal including a bone growth inducing composition which comprises osteogenic protein, insoluble bone matrix and gelatin and a delivery mechanism for delivery of the composition to a treatment site.
  • the osteogenic protein, the insoluble bone matrix and the gelatin may be as hereinbefore described.
  • the delivery mechanism may be a syringe.
  • the composition may be the hydratable powder hereinbefore described which may be contained in the syringe.
  • the material may be hydrated for injection into the delivery site.
  • a method of inducing bone formation in a mammal having a skeletal defect including the step of implanting a bone inducing composition as hereinbefore described into the skeletal defect of the mammal.
  • a method of inducing the growth of ectopic bone in a mammal including the step of implanting a bone inducing composition as hereinbefore described in a non-bony site of the mammal.
  • a method of accelerating allogeneic bone healing in a mammal including the step of implanting allogeneic bone material together with a bone inducing composition as hereinbefore described into site in which allogeneic bone healing in the mammal is required.
  • the allogeneic bone material may be tissue-banked bone including human cortical bone chips, cancellous bone blocks, cancellous bone powder, whole bone or demineralised bone matrix.
  • a method of accelerating autogenous bone graft healing in a mammal including the step of implanting autogenous bone material together with a bone inducing composition as hereinbefore described into site in which autogenous bone graft healing in the mammal is required.
  • the autogenous bone material may be iliac crest autogenous bone.
  • a substance or composition for use in a method of inducing bone formation in a mammal having a skeletal defect the substance or composition comprising a bone growth inducing composition as hereinbefore described and the method including implanting the composition into a skeletal defect of the mammal.
  • a substance or composition for use in a method of inducing the growth of ectopic bone in a mammal the substance or composition comprising a bone growth inducing composition as hereinbefore described and the method including the step of implanting the composition in a non-bony site of the mammal.
  • a substance or composition for accelerating allogeneic bone healing in a mammal comprising a bone growth inducing composition as hereinbefore described and the method including the step of implanting allogeneic bone material together with the composition into a site in which allogeneic bone healing in the mammal is required.
  • the allogeneic bone material may be tissue-banked bone selected from human cortical bone chips, cancellous bone blocks, cancellous bone powder, whole morselised bone or demineralised bone matrix.
  • a substance or composition for accelerating autogenous bone graft healing in a mammal comprising a bone growth inducing composition as hereinbefore described and the method including the step of implanting autogenous bone material together with the composition into a site in which autogenous bone graft healing in the mammal is required.
  • the autogenous bone material may be morselised iliac crest autogenous bone.
  • a substance or composition in the preparation of a medicament for use in a method of inducing bone formation in a mammal having a skeletal defect, the substance or composition comprising a bone growth inducing composition as hereinbefore described.
  • a substance or composition for the preparation of a medicament for use in a method of inducing the growth of ectopic bone in a mammal the substance or composition comprising a bone growth inducing composition as hereinbefore described.
  • a substance or composition in the preparation of a medicament for accelerating allogeneic bone healing in a mammal, the substance or composition comprising a bone growth inducing composition as hereinbefore described and an allogeneic bone material.
  • the allogeneic bone material may be tissue-banked bone selected from human cortical bone chips, cancellous bone blocks, cancellous bone powder, whole bone or demineralised bone matrix.
  • tissue-banked bone selected from human cortical bone chips, cancellous bone blocks, cancellous bone powder, whole bone or demineralised bone matrix.
  • the autogenous bone material may be morselised iliac crest autogenous bone.
  • This invention accordingly relates to the preparation of osteogenic protein from mammalian bone and to its use in conjunction with a matrix in bone repair.
  • BMPs bone morphogenetic proteins
  • the BMPs are redeployed in adults to cause regeneration of bone via mechanisms closely resembling embryonic differentiation.
  • the developmental cascade of bone differentiation consists of chemotaxis of mesenchymal cells, proliferation of progenitor cells, differentiation of cartilage, vascular invasion, bone formation, remodeling, and finally marrow differentiation (Reddi, (1981 ) Collagen Rel. Res. 1 :209- 226). It has been shown that the natural endochondral bone differentiation activity of bone matrix can be dissociatively extracted and reconstituted with inactive residual matrix to restore full bone inductive activity (Sampath and Reddi, (1981 ) Proc. Natl. Acad. Sci. USA 78:7599-7603).
  • European Patent Application No. 148,155 published Oct. 7, 1985, discloses osteogenic proteins derived from bovine, porcine, and human origin.
  • the invention provides osteogenic devices which, when implanted at a skeletal defect site of the mammal, induce at the site of implantation the full regeneration of bone and the consequent healing of the defect.
  • the device comprises a matrix carrier material, as described below, and osteogenic protein, a fraction of total extractable bone protein which contains bone morphogenetic proteins (BMPs).
  • BMPs bone morphogenetic proteins
  • Osteogenic protein requires the presence of a suitable delivery material to exert its bone regenerating effects.
  • Matrix purified from demineralised bone matrix is such a suitable material and is described in more detail below.
  • the method used to isolate osteogenic protein exploits in part, the published procedure of Sampath et al. (1987) (Proc. Natl. Acad. Sci. USA 84, 7109 - 7113.
  • This procedure exploits the BMPs' affinity for heparin and hydroxyapatite immobilized onto chromatographic support matrices to achieve isolation of BMP rich fractions.
  • the procedure entails the chromatography of urea extracts of demineralised bone onto a heparin chromatography column, followed by a hydroxyapatite column, and finally gel exclusion chromatography to eliminate heavy molecular weight contaminants.
  • this procedure results in the effective isolation of a fraction with osteogenic capacity, the quantity, yield and speed of purification of osteogenic protein using the method of the present invention is greatly improved.
  • the preparation of the osteogenic protein of the invention is based on the procedure of Sampath et al (1987), but includes a novel and inventive modification.
  • the key modification involves the fractionation of the total bone protein extract into a high and a low molecular weight fraction at the beginning of the purification process, before chromatography.
  • the bone morphogenetic proteins have a molecular weight of approximately 30 kDa and, for the purposes of this specification, are classed as low molecular weight polypeptides.
  • high molecular weight polypeptides include polypeptides with a molecular weight greater that 100 kDa and especially greater that 300 kDa.
  • the high molecular weight fraction will include collagen and collagen fragments (approximately 100 kDa) as well as collagen aggregates (200 kDa and greater) and other unidentified polypeptides, some of which are thought to be inhibitors of morphogen-induced osteogenesis. It is important to note, for the purposes of this specification, that collagens are separated from the low molecular weight fraction at the beginning of the process before the heparin affinity chromatography step.
  • collagen type I is known to have an affinity for BMPs (Reddi AH (1995) Cartilage morphogenesis: role of bone and cartilage morphogenetic proteins, homeobox genes and extracellular matrix. Matrix Biol. Oct 14(8):599-606. ; Winn SR, Uludag H, Hollinger JO. (1999) Carrier systems for bone morphogenetic proteins. Clin Orthop 1999 Oct (367 Suppl):S95-106).
  • peptides are large MW peptides which tend to foul columns and alter the exchange dynamics of the BMP with the binding sites on the heparin molecule in a way which hampers binding.
  • Figure 1 shows X-ray evaluation scores of treated non-unions as a function of time
  • Figure 2 shows non-union in a bone of a patient after conventional treatment
  • Figure 3 shows complete healing of the bone of the patient of Figure 2 after treatment in accordance with the method of the invention
  • the particulate material was demineralised at room temperature, with consecutive additions of four to five volumes of 0.5 M HCI, until acid base reaction between the hydroxyapatite of the bone and the HCI had neared completion as judged by slowing pH changes over time.
  • the demineralised bone was neutralized with dilute sodium bicarbonate solution, and washed with purified water to produce demineralised bone matrix.
  • Demineralised bone matrix (DBM) from the previous step was extracted twice with three to four volumes of 8M urea, 50mM Tris-HCI, pH 7.4, containing protease inhibitors (5 mM benzamidine hydrochloride, 0.1 M 6-aminohexanoic acid, 5 mM N-ethylmaleimide and 0.5 mM phenylmethylsulfonyfluoride) for 24
  • polypropylene frit and filtered through a three micron nominal size cartridge filter (Polygard, Millipore Corporation, USA).
  • Heavy molecular proteins and collagens were removed by ultrafiltration of the supernatant from the step 2 through a polysulfone 300 kDa nominal molecular weight membrane (Millipore, Cat. No. CDUF006TM).
  • a 100 kDa nominal molecular weight membrane can optionally be employed with somewhat lower yields of total BMP activity, but with higher specific activity.
  • This procedure removed collagens, especially type I collagens, which bind BMPs under conditions of lower ionic strength.
  • the retentate was washed a few times with 6 M urea buffer, 50 mM Tris-HCL pH 7.4 (Buffer A), and the diafiltrate which contained the osteogenic activity was collected.
  • the diafiltrate containing the osteogenic proteins (molecular weight circa 30 kDa) from step 3 was desalted and concentrated by ultrafiltration on a 10 kDa PLGC membrane (Millipore, Cat. No. SK1 P003W4). This step effectively removed salt and other low MW weight components, to create the required conditions for the following chromatographic step. Successive volumes of Buffer A containing the aforementioned concentrations of enzyme inhibitors but excluding n-ethyl maleimide were added to the retentate following concentration, until the conductivity of the retentate reached between 5.0 and 6.0 milli Siemens.
  • the retentate from step 4 was chromatographed onto Heparin-Sephahrose CL- 6b (Pharmacia-Amersham) which had been equilibrated with buffer A containing 0.15 M NaCI.
  • the column was washed with three column volumes of buffer A containing 0.15 NaCI and then eluted with buffer A containing 0.5 M NaCI.
  • the eluting peak with absorbance at 280 nm was collected and stored at 4°C.
  • the retentate from step 6 was chromatographed onto a hydroxyapatite column (Hydroxyapatite Ultrogel, Biosepra, France) which had been equilibrated in Buffer A containing 10 mM sodium phosphate.
  • the column was washed with three column volumes of Buffer A containing 10 mM sodium phosphate.
  • An osteogenic protein enriched fraction was eluted with Buffer A containing 150 mM sodium phosphate.
  • the eluting peak with absorbance at 280 nm was collected and stored at 4°C.
  • the HA affinity fraction from step 7 was exchanged into a 10 mM HCI solution using an Amicon stirred Ultrafiltration cell (Millipore Corporation, U.S.A.) loaded with a 3 kDa cutoff cellulose membrane (YM3, 76 mm regenerated cellulose, lipore Corporation U.S.A.).
  • the HA affinity fraction was instead loaded onto a C-18 Vydac silica-based HPLC column (particle size 5 urn, pore size 300 A).
  • the column was washed with 0.1 % trifluoroacetic acid, 10% acetronitrile for 10 column volumes, and the bound proteins were step eluted with a 70% acetonitrile, 0.1% trifluoroacetic acid. This material was lyophilized and reconstituted into 10 mM HCI.
  • step 8 The material from step 8 was analysed by S-200 gel filtration chromatography (Pharmacia) and found to contain 20% by mass of high molecular weight components. These may be optionally removed by a 'polishing' step employing gel exclusion chromatography on S-200 matrix (Pharmacia) and elution with 8M Urea, 1 M NaCI, 50mM Tris-HCI pH 7.4. The final yield is in the region of 30 mg to 50 mg of osteogenic protein.
  • Osteogenic activity was bioassayed as described by Sampath and Reddi (Proc. Natl. Acad. Sci. USA (1983) 80:6591-6595).
  • the assay consists of implanting test samples comprising insoluble bone matrix and human osteogenic protein in subcutaneous sites in recipient rats under ether anesthesia. A vertical incision (1 cm) was made under sterile conditions in the skin over the thoracic region, and bilateral pockets were prepared by blunt dissection. Implants comprised 25 mg rat ICBM, 50 mg rat tail type I collagen in 0.5 M acetic acid, and osteogenic protein in varying amounts.
  • the test sample was implanted bilaterally into each pocket and the incision was closed with stitches. The heterotropic site allowed for the study of bone induction without the possible ambiguities resulting from the use of bony sites.
  • the implant model in rats exhibited a controlled progression through the stages of osteogenic protein induced endochondral bone development.
  • This postfoetal osteogenesis may be considered to recapitulate events that occur in the normal course of embryonic bone development.
  • the new bone resulted from local mesenchymal condensations, a cartilage phase and extracellular matrix production, vascular invasion and mineralisation, and finally the formation of new bone via the differentiation of osteoprogenitor cell lines.
  • Alkaline phosphatase activity may be used as a marker for osteogenesis.
  • the enzyme activity may be determined spectrophotometrically after homogenization of the implant and assaying of enyme activity with the substrate p-nitrophenyl phosphate under alkaline conditions. Implants showing no bone development by histology should have no alkaline phosphatase activity under these assay conditions (Reddi AH and Sullivan NE (1980) Endocrinology 107, 1291 - 1299).
  • the assay is useful for quantitation of the specific and total activity of alkaline phosphatase, which may then be correlated to the osteoinductive potency of the prepared osteogenic protein described herein.
  • Alkaline phosphatase activity is calculated according to the method of Reddi and Sullivan (1980, Endocrinology 107, 1291-1299). Induction of 1 unit or more of alkaline phosphatase by a rat implant indicates effective osteogenesis.
  • gelatinous solution was cooled to 25°C and 5 volumes of chilled ethanol (-20° C) were
  • the precipitate was dried in vacuo, and milled to a size range of 75 to 425 micron.
  • Human ICBM was used as the adsorptive carrier matrix for the fabrication of the osteoinductive composite biomaterial. Inactive ICBM was restored to biological activity when a sufficient amount of osteogenic protein was combined with ICBM. The particle size of the ICBM influences the quantitative response of new bone. Particles between
  • osteogenic protein in 10 mM HCI and thoroughly mixed with sterile spatula. The material was lyophilized to dryness.
  • This material was then combined with human ICBM-derived gelatin.
  • the components were thoroughly dry-mixed together to obtain a homogeneously distributed composition.
  • the human gelatin acted as a readily hydratable material that causes the biomaterial to become extrudable.
  • the composite was packed into a syringe.
  • the osteoinductive composite was rehydrated at the time of use. Rehydration was achieved when an amount of sterile saline or water was drawn into the syringe, and approximately 10 minutes allowed for rehydration to occur.
  • the material could then easily be expelled out of the syringe by depressing the plunger. This allowed for precise implant deposition into a defect site at the time of surgery.
  • the implant may be further contained in situ using standard gelatinous sponges such as Spongostan (Johnson and Johnson Medical Limited, U.K.).
  • the carrier could be replaced by either a biodegradable-synthetic or synthetic- inorganic matrix (e.g., HAP, collagen, tricalcium phosphate, or polylactic acid, polyglycolic acid and various copolymers thereof).
  • a biodegradable-synthetic or synthetic- inorganic matrix e.g., HAP, collagen, tricalcium phosphate, or polylactic acid, polyglycolic acid and various copolymers thereof.
  • Table 3 sets out a typical formulation for the osteogenic composition.
  • Implants containing 500 micrograms of human osteogenic protein adsorbed onto a composite matrix comprising 1g of insoluble bone matrix and 200 mg of lyophilized human gelatin were prepared. Thirty-four patients with resistant nonunions including partial or complete segmental defects were treated with the osteogenic composite. The series consisted of 11 females and 23 males. The average age was 36 years. All patients had previously been variously treated by internal or external fixation, cast, and/or autogeneic bone grafting, and failed to achieve union. Preoperative symptoms averaged 26 months (range, one to 228 months). The implant was incorporated at the time of surgery by injecting the hydrated implant at the defect site, which was further stabilised by internal or external fixation. An average of 2.4g of the composite was used per patient.
  • the multistep developmental cascade of bone induced by the osteogenic biomaterial composite of the invention includes binding of fibrin and fibronectin to the biomaterial, chemotaxis of cells, proliferation of fibroblasts, mesenchymal condensation, differentiation into chondroblasts, chondrogenesis, vascular invasion, bone formation, remodeling, and bone marrow differentiation.
  • the injectable biomaterial of the invention offers several advantages. It may be stored at room temperature for lengthy periods without marked deterioration in biological activity. It may be readily rehydrated at the time of surgery and it is easily handled, merely requiring the depression of the syringe plunger to expel the osteogenic material as required.
  • the osteogenic biomaterial composite offers the following advantages. It is osteogenic, inducing bone at the site of implantation. It obviates the need to perform a second operation at the patient's hip to harvest autologous bone and it obviates the need to use tissue banked bone. This reduces the risk of transmissible diseases.
  • the ICBM binds osteogenic protein and acts as a slow release delivery system to activate progenitor cells at the site of implantation.
  • the composite biomaterial of the invention accommodates each step of the cellular response during bone development. It is biocompatible, and is resorbed during osteogenesis and replaced by the host's own bone.
  • the geometry of the described biomaterial as measured by its particle size, is optimal in permitting cell infiltration and differentiation.

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  • Oral & Maxillofacial Surgery (AREA)
  • Dermatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Immunology (AREA)
  • Toxicology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Materials For Medical Uses (AREA)
  • Peptides Or Proteins (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

Dans une méthode permettant d’isoler une protéine ostéogène d’os, duquel une fraction contenant une protéine ostéogène est extraite et enrichie au moyen d’une séquence d’étapes d’enrichissement sélectionnées dans le groupe formé par l’ultrafiltration et la chromatographie, l’invention concerne la suppression améliorée de composants de poids moléculaire élevés de la fraction contenant une protéine ostéogène avant les étapes d’enrichissement. Les composants de poids moléculaire élevé possèdent un poids moléculaire d’environ 100 - 300 kDa et sont sélectionnés dans le groupe constitué par le collagène, des fragments de collagène, des agrégats de collagène et des mélanges de ceux-ci.
PCT/IB2003/002354 2002-06-20 2003-06-18 Biomateriaux osteo-inducteurs Ceased WO2004004630A2 (fr)

Priority Applications (7)

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AU2003240196A AU2003240196A1 (en) 2002-06-20 2003-06-18 Osteoinductive biomaterials
JP2004519039A JP2005536489A (ja) 2002-06-20 2003-06-18 骨誘導性の生体材料
EP03732811A EP1539812B1 (fr) 2002-06-20 2003-06-18 Biomateriaux osteo-inducteurs
AT03732811T ATE469916T1 (de) 2002-06-20 2003-06-18 Osteoinduktive biomaterialien
CA002527619A CA2527619A1 (fr) 2002-06-20 2003-06-18 Biomateriaux osteo-inducteurs
DE60332848T DE60332848D1 (de) 2002-06-20 2003-06-18 Osteoinduktive biomaterialien
US10/518,723 US7728116B2 (en) 2002-06-20 2003-06-18 Method of preparing an osteogenic protein fraction

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ZA2002/4977 2002-06-20
ZA200204977 2002-06-20

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AT (1) ATE469916T1 (fr)
AU (1) AU2003240196A1 (fr)
CA (1) CA2527619A1 (fr)
DE (1) DE60332848D1 (fr)
WO (1) WO2004004630A2 (fr)

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JP2007526085A (ja) * 2004-03-02 2007-09-13 ナノセラピューティクス・インコーポレイテッド 骨を修復するための組成物ならびにそのような組成物を調製および使用するための方法
CN100377741C (zh) * 2004-02-23 2008-04-02 江卫世 注射用复方骨肽及其制备工艺
AU2005328325B2 (en) * 2004-11-29 2012-04-05 Department Of Veterans Affairs Activating extraction of demineralized bone matrix
US8415302B2 (en) 2004-01-28 2013-04-09 The Regents Of The University Of California Surgical applications for BMP binding protein
US8759296B2 (en) 2004-01-28 2014-06-24 The Regents Of The University Of California Bone morphogenic protein binding peptide
US8975231B2 (en) 2004-01-28 2015-03-10 The Regents Of The University Of California Bone morphogenic protein binding peptide
US9072709B2 (en) 2009-06-23 2015-07-07 The Regents Of The University Of California Enhancement of bone morphogenic protein (BMP) retention with BMP binding peptide (BBP)

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JP2009120449A (ja) * 2007-11-16 2009-06-04 Amino Up Chemical Co Ltd バイオアパタイトの製造方法及び生物学的な活性を有する物質の分離方法
US8907059B2 (en) * 2008-11-14 2014-12-09 Bio-Rad Laboratories, Inc. Phosphopeptide enrichment of compositions by fractionation on ceramic hydroxyapatite
ES2552668T3 (es) 2010-06-28 2015-12-01 Bbs - Bioactive Bone Substitutes Oy Una preparación de proteína ósea en una matriz de polietilenglicol/glicerol
KR101531479B1 (ko) * 2014-11-21 2015-06-26 세원셀론텍(주) 의료용 재료로 사용하기 위한 고농도 콜라겐 제조방법

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US8975231B2 (en) 2004-01-28 2015-03-10 The Regents Of The University Of California Bone morphogenic protein binding peptide
US8415302B2 (en) 2004-01-28 2013-04-09 The Regents Of The University Of California Surgical applications for BMP binding protein
US8759296B2 (en) 2004-01-28 2014-06-24 The Regents Of The University Of California Bone morphogenic protein binding peptide
US9050300B2 (en) 2004-01-28 2015-06-09 The Regents Of The University Of California Surgical applications for BMP binding protein
US9333237B2 (en) 2004-01-28 2016-05-10 The United States of America National Institutes of Health (NIH), U.S. Dept of Health and Human Services (DHHS) Bone morphogenic protein binding peptide
US9610320B2 (en) 2004-01-28 2017-04-04 Regents Of The University Of California Surgical applications for BMP binding protein
US9855368B2 (en) 2004-01-28 2018-01-02 The Regents Of The University Of California Bone morphogenic protein binding peptide
CN100377741C (zh) * 2004-02-23 2008-04-02 江卫世 注射用复方骨肽及其制备工艺
JP2007526085A (ja) * 2004-03-02 2007-09-13 ナノセラピューティクス・インコーポレイテッド 骨を修復するための組成物ならびにそのような組成物を調製および使用するための方法
AU2005328325B2 (en) * 2004-11-29 2012-04-05 Department Of Veterans Affairs Activating extraction of demineralized bone matrix
US8753660B2 (en) 2004-11-29 2014-06-17 The Regents Of The University Of California Activating extraction of demineralized bone matrix
US9072709B2 (en) 2009-06-23 2015-07-07 The Regents Of The University Of California Enhancement of bone morphogenic protein (BMP) retention with BMP binding peptide (BBP)
US9694047B2 (en) 2009-06-23 2017-07-04 The Regents Of The University Of California Enhancement of bone morphogenic protein (BMP) retention with BMP binding peptide (BBP)

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DE60332848D1 (de) 2010-07-15
CN1678631A (zh) 2005-10-05
AU2003240196A1 (en) 2004-01-23
EP1539812B1 (fr) 2010-06-02
JP2005536489A (ja) 2005-12-02
EP1539812A2 (fr) 2005-06-15
ATE469916T1 (de) 2010-06-15
WO2004004630A3 (fr) 2004-08-19
US20060093640A1 (en) 2006-05-04
CA2527619A1 (fr) 2004-01-15
AU2003240196A8 (en) 2004-01-23
US7728116B2 (en) 2010-06-01

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